Human chorionic gonadotropin (hCG) is a glycoprotein hormone produced during pregnancy. The hCG hormone is produced by the developing embryo soon after fertilization and later by syncytiotrophoblast cells in the developing placenta, and its primary function is to maintain the production of progesterone by the corpus luteum in early pregnancy. The hCG hormone is a member of the glycoprotein hormone family (GPH) that also includes luteinizing hormone (LH), follicle stimulating hormone (FSH), and thyroid stimulating hormone (TSH). All members of the GPH family are heterodimers that consist of an alpha and beta subunit. The alpha subunit is conserved across the GPHs, while the beta subunit of hCG is unique, although there remains about 80% homology across the beta subunits within the GPH family. In addition to conferring differentiation, the beta subunit also grants each heterodimer its unique biological activity and receptor specificity.
Intact hCG, which is also known as regular hCG, has a molecular weight of around 37 kDa and is composed of 244 amino acids. The alpha subunit contains 92 amino acids and the beta subunit contains 145 amino acids. Intact hCG is generally present at varying levels throughout pregnancy, has been noted to be the predominant form of hCG in later stages of pregnancy, and has previously been believed to have the most biological relevance throughout all stages of pregnancy. Multiple variants of hCG that exhibit independent functions are currently recognized and one of these is hyperglycosylated hCG (hCG-H) which is predominantly produced in early pregnancy. A variety of dissociation and degradation products of hCG are found in blood and urine and these include, among others, nicked hyperglycosylated hCG, nicked hCG, free hCG beta subunit (hCG-β), nicked free hCG beta subunit, nicked hyperglycosylated hCG free beta subunit, and hCG beta core fragment (hCG-βcf). See de Medeiros et al., Human Reproduction Update 15(1): 69-95 (2009) and Birken et al., Endocrinology 123: 572-583 (1988), which are incorporated herein by reference.
In early pregnancy urine, the levels of hCG and its dissociation and degradation products, collectively referred to as hCG isoforms or variants, vary greatly from day to day and from subject to subject. Most pregnancy tests have been developed to preferentially detect intact hCG or to detect some combination of hCG, hCG-H, and hCG-β.
One distinct degradation product of hCG is the hCG beta core fragment (hCG-βcf), which is a fragment of hCG-β, has a molecular weight of about 14 kDa, and contains 73 amino acids in its protein core. The hCG-βcf isoform is now recognized to be a predominant form of hCG present in urine from about seven weeks of pregnancy. See de Medeiros et al., Obstet. Gynecol, 80(2): 223-230 (1992), which is incorporated herein by reference. It has been noted that high levels of hCG-βcf can cause false negative results in urine-based pregnancy tests. For more information, see Diwan, Med. Lab. Observer pg. 18-20 (February 2011); Cervinski et al., Clin. Chem. Lab. Med. 48(7): 935-942 (2010); Gronowski et al., Clin. Chem. 55(7): 1389-1394 (2009), which are incorporated herein by reference. Previous and current pregnancy tests, however, do not detect this isoform or have only limited detection capability for this isoform in urine samples.
Accurate and rapid detection of low levels of various hCG isoforms is desirable to confirm pregnancy early after conception has occurred. Further, it is desirable to minimize incidences of false negative results. Accordingly, it would be beneficial to develop a pregnancy test to detect multiple relevant forms of hCG (e.g., all clinically relevant forms of hCG) to increase the reliability and accuracy of pregnancy detection, particularly in early pregnancy.